Aberrant Chromatin Remodeling by Retinoic Acid Receptor Fusion Proteins Assessed at the Single-Cell Level

Supplemental Material

This article contains the following supporting material:

• Supplemental Figure 1 - Ability of wild-type LacR-RARα and LacR-X-RARα to form hetero-oligomer or homo-oligomer complexes on the lac operator array within A03_1 cells. (A) A03_1 cells were transiently transfected YFP-RXR aloneα (bottom row) or co-transfected with YFP-RXRα and CFP-LacR-RARα (first row), CFP-LacR-PML-RARα (second row), CFP-LacR-PLZF-RARα (third row) and were subjected to deconvolution microscopic analysis. (B-D) A03_1 cells were co-transfected with YFP-RARα and CFP-LacR-RARα (B, top row), YFP-PML-RARα and CFP-LacR-PML-RARα (C, top row), YFP-PLZF-RARα and CFP-LacR-PLZF-RARα (D, top row) or transfected with YFP-RARα alone (B, bottom row), YFP-PML-RARα alone (C, bottom row) or YFP-PLZF-RARα alone (D, bottom row) and subjected to deconvolution microscopic analysis. Nuclei were visualized by DNA staining with DAPI. Images shown are representative of the nuclei detected using the CFP or YFP filter (first column), DAPI filter (second column) or the results of merging the signals from the first two columns (third column).
• Supplemental Figure 2 - Effect of CFP-LacR-RARα or CFP-LacR-X-RARα binding on the lac operator array area within RRE_B1 cells. (A) RRE_B1 cells were transiently transfected with CFP-LacR-tagged wide-type RARα or X-RARα and incubated with or without ATRA (10^-6 M for three hours). The area of the arrays was determined as described in the Material and Methods section and presented as the geometric mean ± 95% confidence interval (CI). (B) Representative images are shown for arrays bound by CFP-LacR (first row), CFP-LacR-RARα (second row), CFP-LacR-PML-RARα (third row) and CFP-LacR-PLZF-RARα (bottom row) within RRE_B1 cells incubated without ATRA (left column) and with ATRA (right column). The number of cells examined, the geometric mean (GM) and a histogram showing the distribution of array area is presented below each representative image. Scale bar, 2 μm.
• Supplemental Figure 3 - Effect of ATRA on protein stability of CFP-LacR-RARα and CFP-LacR-X-RARα . A03_1 cells were transiently transfected with CFP-LacR-RARα , CFP-LacR-PML-RARα and CFP-LacR-PLZF-RARα constructs as indicated and incubated without (-) or with (+) ATRA (10^-6 M for 3 hours) as indicated. The proteins from whole-cell extracts of transiently transfected A03_1 cells were separated by 7.5% SDS-PAGE and immunoblotted with GFP antibody.
• Supplemental Figure 4 - Effect of wild type RARα and X-RARα on the localization and distribution of co-activator SRC-1 and co-repressor SMRT. A03_1 cells were transiently co-transfected with CFP-LacR-RARα (A), CFP-LacR-PML-RARα (B), CFP-LacR-PLZF-RARα (C) plus YFP-SRC-1 and ChFP-SMRT and incubated in the absence (top row) or presence (bottom row) of retinoic acid (10^-6 M ATRA for three hours) then subjected to deconvolution microscopic analysis. (D) A03_1 cells were transiently transfected with YFP-SRC-1 (top row) or ChFP-SMRT (bottom row) alone and subjected to analysis with deconvolution microscopy. Nuclei were visualized by DNA staining with DAPI. Images shown are representative of the nuclei detected using the filter indicted (first to penultimate column) or represent the results of merging the signals from the first two-to-four columns (last column).